The enzyme gamma glutamyltransferase (GGT) catalyses the transfer of a gamma glutamyl moiety to an amino acid or peptide. The enzyme is present in the liver, kidney, prostate, pancreas, blood and other tissues. GGT is believed to be part of the glutathione cycle.
Elevation of the blood GGT level has been demonstrated to be an indicator of various diseases. Initially, the elevation of the blood GGT level, as measured by increased GGT activity in blood samples, was used as a test for diseases of the liver, bile ducts, and pancreas. It has since become commonly used as a general indicator of liver disorder. Increased blood GGT activity is indicative of liver dysfunction and may indicate pathology due to high alcohol consumption, drug use or disease. Elevated GGT activity has also been noted in conjunction with other diseases such as myocardial infarction, pancreatitis, and intra-cerebral tumors.
Currently, the amount of GGT present in blood is commonly measured using a kinetic assay on blood serum wherein (gamma-L-glutamyl)-p-nitroanilide and glycylglycine are the substrates for the GGT enzymatic formation of p-nitroaniline. The substrates are allowed to react with serum GGT for approximately 10 minutes. The p-nitroaniline produced in this reaction is then measured spectrophotometrically in the wavelength range of 405-410 nm. The rate of p-nitroaniline formation is proportional to GGT activity. Therefore, a high conversion to p-nitroaniline is indicative of a high GGT concentration in the serum, which may be due to liver degradation.
This method, however, is not applicable to analysis of blood samples in which the red blood cells have been ruptured or hemolysed. Any disruption of red blood cells prior to testing will interfere with an accurate determination of GGT activity, due to the strong light absorption by hemoglobin in the wavelength range used to observe the formation of p-nitroaniline. Methods of analysis have been developed to deal with the presence of hemoglobin in the sample. These methods involve post-reaction precipitation of blood protein by trichloroacetic acid followed by centrifugation of the sample. While this procedure avoids hemoglobin interference it retains the need for a large fluid blood sample volume that must be obtained from the subject and adds additional steps to the assay procedure.
An alternative to direct measurement of the p-nitroaniline is a method in which the GGT enzyme reaction product, p-nitroaniline, is diazotized and coupled with a chromophore to produce a mixture more suitable for spectrophotometric analysis. This method, however, utilizes blood serum and is not applicable to whole blood samples or samples of dried blood as is the method of the present invention.
Presently available assay methods which utilize fluid blood serum samples for determination of GGT activity are ineffective when blood sample damage has occurred during shipping. Sample damage may result in hemolysis or enzyme denaturation with subsequent enzyme activity loss. In addition, leaking fluid sample packages create a potential health hazard for personnel who are required to handle packages containing fluid blood, physiological fluids, or chemical reagents.
Analysis of GGT activity in blood is being employed by insurers to determine the desirability of offering insurance contracts to potential customers. This use is increasing the submission of physiological fluid samples, such as blood or urine, to laboratories from persons residing far from the laboratory. The advent of such distant sampling presents a number of problems in assaying for GGT activity. Conveyance of these samples to the laboratory often occurs under poor conditions. Mistreatment of blood samples and concomitant rupture of red blood cells introduces hemoglobin into the sample resulting in interference with the current GGT assay procedures. The use of public and private mail delivery systems to transport such samples increases the potential for careless handling. Damaged and leaking fluid samples may alarm or endanger mail system workers who contact the damaged and leaking packages of blood or other fluid physiological materials. Finally, mishandling of blood samples may cause a loss of GGT activity due to denaturation of the GGT enzyme and result in unreliable test results.